Identification, expression and functional characterisation of CYP1A in grass carp (Ctenopharyngodon idella)

Highlights

• The grass carp (Ctenopharyngodon idella) CYP1A was identified and analysed.

• Tissue distribution of CiCYP1A and the expression pattern in response to GCRV infection were analysed.

• CiCYP1A suppressed the promoter activity of IFN-I signaling pathway genes.

• CiCYP1A suppressed mRNA expression levels of IFN-I.

Abstract

In mammal, CYP1A has attracted special attention due to its important roles in the oxidative metabolism. In fish, the researches on CYP1A are more focus on its roles in pollution in water environments, but the immune function is unclear. In the study, CiCYP1A gene was cloned from grass carp (Ctenopharyngodon idella). Tissue distribution exhibited an overwhelmingly high basal expression levels in the liver. After GCRV infection, CiCYP1A showed a potent response, indicating CiCYP1A was involved in GCRV-induced immunity. Subcellular localisation showed CiCYP1A was distributed in the cytoplasm. Besides, dual-luciferase activity assays revealed CYP1A was relevant for IFN-I signaling pathway modulation, furthermore, overexpressed CYP1A potently suppressed the mRNA expression of IRF3 and IFN-I but not IRF7. The results provide new sights into exploring immune function of CiCYP1A in teleosts.

Introduction

Grass carp (Ctenopharyngodon idella) is a native economical aquaculture species in China, accounting for approximately 10% of total aquaculture production in 2017 [1], which contributes a lot to meet the growing demand for aquatic animal protein. However, the frequent outbreak of grass carp hemorrhagic disease has brought huge losses to aquaculture industry [2]. Grass carp hemorrhagic disease was caused by grass carp reovirus (GCRV), the GCRV-infected fish would initiate intense immune responses, such as defense response, inflammatory response, and innate immune response to resist invaders [[3], [4], [5]], but GCRV can still escape the host's immune system by blunting the transcription of host interferon (IFN) [6]. Currently, although the pathogenesis of hemorrhagic disease has not been well revealed, the completion of draft genome of grass carp provides great hope for breeding new disease-resistant varieties, and also helps to explore the gene function of fish [7,8].

Cytochrome P450 enzymes (CYPs) are a group of heme-thiolate monooxygenases, which are involved in the oxidative metabolism of endogenous compounds and xenobiotics, thereby playing critical roles in cholesterol and hormone synthesis, the synthesis and degradation of biogenic amines, synthesis and metabolism of vitamin D₃, the hydroxylation of retinoic acid and presumably other morphogens, drug deactivation, and xenobiotics detoxification [[9], [10], [11], [12], [13], [14]]. Mammalian CYP enzymes consist of 57 genes, which are classified into 18 families according to their cDNA and amino acid sequence identities [[15], [16], [17]]. Over the past decade, the CYP families have been relatively well studied as its function of biotransformation reactions with activation of prodrugs or degradation of exogenous substances in the liver. Notably, the researches on the CYPs have recently become the subject of particular scientific interest because CYPs are involved in various diseases including cancer and immune regulation [[18], [19], [20], [21], [22]].

Among the numerous of CYP family members, CYP1A has attracted special attention due to its important roles in metabolically activating many environmental procarcinogens in the polyaromatic hydrocarbons (PAHs), polyhalogenated aromatic hydrocarbons (PHAHs), aryl- and alkyl-amine, heterocyclic amine, and dioxins [9,23]. In view of this, CYP1A is also extensively used as a biomarker to assess contamination of the aquatic environment [[24], [25], [26], [27]]. CYP1A is regulated by the aryl hydrocarbon receptor (AHR), which can interact with nuclear factor κB (NFκB), oestrogen receptor-α (ESRα) and retinoblastoma protein 1 (RB1), leading to the transcription of genes involved in growth, cell cycle, apoptosis, and immunity [22,[28], [29], [30]]. In fish, a large number of researches about CYP1A are focus on its roles in pollution in water environments [[31], [32], [33], [34]]. However, little studies about CYP1A on immunity were reported. In this study, CYP1A was identified from Ctenopharyngodon idella (named as CiCYP1A) and functionally characterised. The expression profiles of CiCYP1A and subcellular localisation were analysed. Besides, the regulation of CiCYP1A on IFN-I signaling pathway was explored. The results provided further insights into the gene function of CiCYP1A in teleost.